1. Field of the Invention
The present invention relates generally to the fields of biochemical endocrinology and regulation of bone formation and degradation. More specifically, the present invention relates to the regulation of osteoblast function by the osteoclast secreted chemokine-like protein mim-1 and uses thereof.
2. Description of the Related Art
Osteoclasts are multinucleated cells formed by fusion of precursors derived from pleuripotential hematopoietic stem cells (1) that circulate in the monocyte fraction (2, 3). Differentiation of the precursors into osteoclasts is a complex process that requires both M-CSF and RANKL (ODF, osteoclast differentiation factor; also known as TRANCE) (4, 5). The mechanism(s) by which osteoclastic precursors are recruited to an area of bone resorption, establish and differentiate into mature osteoclasts is a complex process that is still not fully understood.
Mature osteoclasts are terminally differentiated cells and while it is clear that M-CSF and RANKL are essential for differentiation of osteoclasts, additional osteoclast-inductive agents or synergistic effectors of RANKL are likely to be important in the development of active mature osteoclasts (6, 7). In fact, RANKL/TRANCE is not bone-specific since it was first cloned as a tumor necrosis factor (TNF) related activation-induced cytokine (TRANCE) in T-cell hybridomas suggesting a potential role in immune function (8).
Communication, via a variety of signaling molecules, has long been proposed as a key component in the homeostatic signaling process between osteoclasts and osteoblasts (9, 10). Osteoclasts respond to numerous factors that are derived from bone or the bone microenvironment including, among others, IL-1, IL-6, TNF-xcex1 and TGF-xcex2 and osteoprotegrin (6, 7, 10-13). Under conditions of normal bone turnover, bone resorption is followed by new bone synthesis. The mechanisms regulating recruitment of osteoblast precursors into areas recently degraded are poorly understood, but presumably involve a signaling pathway between osteoclasts and osteoblasts (14).
The prior art is deficient in methods of regulating the secretion of a chemokine-like protein expressed specifically by cells of hematopoietic origin, like osteoclasts, so as to manipulate a signaling pathway that may be involved in regulating recruitment of osteoblast precursor cells to areas of recent bone resorption b y osteoclasts. The present invention fulfills this long-standing need and desire in the art.
Mim-1 is a protein reported to be expressed specifically by cells of hematopoietic origin (15), which includes osteoclasts. Mim-1 is distinct from, but homologous with, the neutrophil chemokine protein, LECT2, and is an abundant protein in osteoclasts. In addition, mim-1 is secreted in a time dependent manner in vitro. Furthermore, secretion of mim-1 is stimulated in a PMA concentration dependent manner. Secretion of mim-1 precedes the largest increase in PMA stimulated bone resorption by isolated osteoclasts. Immunofluorescence microscopy demonstrated that both avian osteoclasts and human osteoclast-like cells but not mesenchymal stem cells (which includes osteoblast precursors) express mim-1. Mim-1 may be a key signaling protein secreted by osteoclasts that regulates recruitment and/or differentiation of osteoblast precursor cells, thereby providing an essential mechanism for regulating the mass and structural integrity of bone.
The present invention is drawn to methods of inducing recruitment and proliferation of osteoblasts, increased bone resorption by osteoclasts and decreased differentiation of osteoclast precursor cells following secretion or administration of mim-1. Generally, the mim-1 protein has the sequence of SEQ ID NO. 8 or a fragment thereof.
In another aspect of the present invention, there is provided methods of inducing bone resorption activity of osteoclasts, inducing recruitment and proliferation of osteoblasts, and inducing new bone synthesis in an individual by mim-1 protein. Generally, the mim-1 protein has the sequence of SEQ ID NO. 8 or a fragment thereof.
In yet another aspect of the present invention, there is provided a method of stimulating bone marrow cell differentiation in an individual by a mim-1 protein. Generally, the mim-1 protein has the sequence of SEQ ID NO. 8 or a fragment thereof.
In one more aspect of this invention, there is provided a pharmaceutical composition comprising mim-1 protein in a pharmaceutically acceptable vehicle. Generally, the mim-1 protein has the sequence of SEQ ID NO. 8 or a fragment thereof.
In a further aspect of the present invention, there is provided a method of inhibiting cancer cell growth in an individual with a pharmaceutical composition comprising mim-1 protein in a pharmaceutically acceptable vehicle, where mim-1 protein generally has the sequence of SEQ ID NO. 8 or a fragment thereof.
In a still further aspect of the present invention, there is provided a polyclonal antibody against and specifically binds to mim-1 protein, a pharmaceutical composition comprising the polyclonal antibody in a pharmaceutically acceptable vehicle and a method to inhibit excessive bone synthesis in an individual with the pharmaceutical composition comprising the polyclonal antibody in a pharmaceutically acceptable vehicle. Generally, mim-1 protein generally has the sequence of SEQ ID NO. 8 or a fragment thereof.
In yet a further aspect of the invention, there is provided a recombinant mim-1 gene which produces mim-1 antisense mRNA that hybridizes to endogenous mim-1 mRNA and inhibiting expression of endogenous mim-1, a vector expressing recombinant mim-1 gene, a host cell comprising the vector expressing recombinant mim-1 gene and a method to inhibit excessive bone synthesis in an individual with the vector expressing recombinant mim-1 gene.
Other and further aspects, features, and advantages of the present invention will be apparent from the following description of the presently preferred embodiments of the invention. These embodiments are given for the purpose of disclosure.